Simultaneous determination of testosterone metabolites in liver microsomesusing column-switching semi-microcolumn high-performance liquid chromatography

A sensitive and selective column-switching semi-microcolumn high-performanc e liquid chromatographic (HPLC) method has been developed for the simultane ous determination of testosterone and eight of its metabolites (6 alpha-, 6 beta-, 16 alpha-, 16 beta-, 7 alpha-, 2 alpha-, and 2 beta -hydroxytestost erone, and androstenedione) in liver microsomes. After incubation for 10 mi n, testosterone and its metabolites were extracted from the microsomes with ethyl acetate, and the extract was evaporated to dryness. The residue was dissolved in the mobile phase and loaded onto the HPLC system. The analytes were first concentrated in a precolumn and subsequently transferred to the analytical column, where they were separated using linear gradient elution . A UV detector set at 254 nm was used to detect the analytes. This newly d eveloped method clearly separated TES and the metabolites with high resolut ion and was found to be reproducible with intra- and interday variability o f < 10.7%. This method has been subsequently used to determine the testoste rone hydroxylation activities catalyzed by 15 different recombinant CYP iso zymes. The results confirmed the formation of stereoselectively hydroxylate d metabolites by each CYP isozyme. (C) 2001 Academic Press.